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Cell Proliferation Activities on Skin Fibroblasts from a Short Child with Absence of One Copy of the Type 1 Insulin-Like Growth Factor Receptor (IGF1R) Gene and a Tall Child with Three Copies of the IGF1R Gene

Departments of Pediatrics (Y.O., C.J.P., T.S., D.B.D.), Clinical Biochemistry (K.S., S.O.), and Medical Genetics (H.F., L.W.), University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom; Clinical and Molecular Genetics Unit (L.C.W.), Department of Animal Resource Sciences and Applied Biological Chemistry (T.F., S.-I.T.), Graduate School of Agriculture and Life Sciences, The University of Tokyo, 113-8657 Tokyo, Japan; and Biochemistry, Endocrinology and Metabolism Unit (R.S.), Institute of Child Health and Great Ormond Street Hospital, 30 Guilford Street, London WC1N 1EH, United Kingdom

Address all correspondence and requests for reprints to: David B. Dunger, M.D., University of Cambridge, Addenbrooke’s Hospital, Level 8, Box 116, Hills Road, Cambridge CB2 2QQ, United Kingdom.

	Abstract

Top Abstract Introduction Clinical Report Materials and Methods Results Discussion References

 
The type 1 IGF receptor (IGF1R) is required for normal embryonic and postnatal growth. The aim of this study was to determine whether we could detect abnormal IGF1R function in skin fibroblasts from children with an abnormal copy number of the IGF1R gene.

We report two children with altered copy number of the IGF1R gene who presented with abnormal growth. Case 1 is a girl with intrauterine growth retardation, postnatal growth failure, and recurrent hypoglycemia. Pituitary function tests were normal. Routine karyotype analysis identified a deletion on 15q26.2, and a fluorescence in situ hybridization study using IGF1R probes showed only a single IGF1R gene. Case 2 was large for gestational age, with birth weight and length at or above 97th percentile, and showed rapid early postnatal growth. He was found to have a recombinant chromosome 15 containing a partial duplication at 15q (q25-qter). A fluorescence in situ hybridization study using the same probes showed three copies of the IGF1R gene.

In a mitochondrial activity assay, skin fibroblasts from the subject with only one copy of IGF1R showed slower growth, whereas cells from the subject with three copies of IGF1R showed accelerated growth compared with controls. IGF1R phosphorylation, as assessed by Western blot, and IGF1R binding studies were decreased compared with controls in the child with one copy of the IGF1R and increased in the child with three copies of the gene. Our data are consistent with the concept that IGF1R gene copy number is of functional and clinical importance in humans.

	Introduction

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INSULIN-LIKE GROWTH FACTORS (IGFs) and their receptors regulate embryonic and postnatal growth (1, 2). Genetic evidence derived from targeted mouse mutants indicates that both the insulin receptor (IR) and type 1 IGF receptor (IGF1R) are required for normal embryonic growth (3, 4). In the earlier embryonic phase, fetal growth is largely IGF-II-dependent through IGF1R and IR (5), whereas later in fetal life and postnatally IGF-I is dominant in growth regulation.

In animals heterozygous for a classical targeted disruption of IGF1R gene, normal levels of receptor mRNA and normal growth were observed, suggesting effective up-regulation of the second (intact, wild-type) allele and that a single functional wild-type allele of the IGF receptor is sufficient to assure normal expression and growth (4). Because the classical homozygous IGF1R knock-out mice die at birth of respiratory failure, to assess the postnatal role of IGF1R, a conditional IGF1R gene-targeted model was recently established (6, 7). Not only homozygous mice, but also heterozygous mice showed postnatal growth failure.

In humans, the IGF1R is the product of a single-copy gene located at bands q26.3, at the distal end of chromosome 15 (8). The IGF1R is an 2ß2-heterotetrameric protein with ligand-stimulated tyrosine kinase activity (9). Binding of IGF-I to its receptor induces receptor autophosphorylation in the intracellular kinase domain of the ß-subunit and results in activation of the intrinsic tyrosine kinase activity of the IGF1R.

There have been several cases of intrauterine growth retardation (IUGR) in patients with deletion of the distal long arm of chromosome 15 or ring chromosome 15, which had resulted in the loss of one copy of the IGF1R gene but normal levels of IGF-I (10). Recently, a case with IUGR and postnatal growth retardation was reported in which the patient had high IGF-I levels and mutations that could alter the ligand binding domain of the IGF1R and induce IGF-I resistance (11). However, the functional significance of loss or gain in IGF1R expression has not been elucidated.

To explore these questions, we have investigated the IGF-I stimulated cell proliferation rate and autophosphorylation of the IGF1R ß-subunit in two children with altered copy number of IGF1R gene and normal controls.

	Clinical Report

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Case 1 is a 10-yr-old girl with short stature. She was born at term after an uneventful pregnancy, and her birth weight was 2660 g (2nd percentile). She has triangular facies (Fig. 1A) with upward slanting palpebral fissures, blepharophimosis, a relatively prominent nose with a short philtrum, bilateral clinodactyly of the fifth finger, very flat feet, hyperextensible knees, limited pronation/supination at the elbows, and a single cafe-au-lait patch on her abdomen. Heights of her father and mother are 156 and 160 cm, respectively, and her 14-yr-old brother is healthy and normally grown. At the age of 1 month, she was investigated for IUGR and subsequent failure to thrive, but no significant abnormalities were found, except for an atrial septal defect and ventricular septal defect that were initially managed conservatively. She continued to grow poorly and she underwent surgery for repair of the cardiac septal defects at the age of 9 months. She showed improved growth after the operation although she was left with a residual ventricular septal defect. In the follow-up, however, her height and weight remained below the 3rd percentile (Fig. 2A), and her head circumference was between 3rd and 10th percentiles. She also developed recurrent symptoms of hypoglycemia. Consequently, she was admitted for assessment of pituitary function at the age of 22 months. A 3-h glucagon test showed adequate GH (basal, 8.3 mU/liter; stimulated, 30.4 mU/liter) and cortisol (basal, 397 nmol/liter; stimulated, 954 nmol/liter) responses. Her blood glucose after an overnight fast was 1.1 mmol/liter, and it remained low (between 1.0 and 1.3 mmol/liter) during the glucagon test. Head magnetic resonance imaging was not suggestive of any pituitary abnormalities. GH replacement therapy was initiated mainly to prevent hypoglycemia (1.7 U/d). When she was 2 yr and 9 months old, her 24-h blood glucose profile indicated persisting hypoglycemia (glucose, 1.9 mmol/liter), and she had some spontaneous endogenous GH secretion despite exogenous GH therapy. Some catch-up growth was subsequently observed, with maximum height velocity of 9.9 cm/yr, but this has leveled off, and during the last 4 yr she has been growing steadily on the 3rd height percentile. At 10 yr she remains short (0.4th to 3rd percentile) and thin (2nd to 9th percentile). Her age-adjusted weight has slowly increased. Her development of speech and motor skills was moderately retarded during the first years of life, but later she has caught up to some extent and now attends mainstream school despite some learning difficulties.

View larger version (76K): [in this window] [in a new window]   FIG. 1. Dysmorphic features of the two cases. A, Case 1. Triangular facies, upslanting palpebral fissures, blepharophimosis, prominent nose. B and C, Case 2. Malar hypoplasia, small ears with thickened helix, prominent chin.

View larger version (19K): [in this window] [in a new window]   FIG. 2. Growth charts for the two cases. A, Case 1. The starting point of GH therapy is indicated with an arrow. B, Case 2.

	Materials and Methods

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Materials

Recombinant human IGF-I (rhIGF-I) was kindly provided by CHIRON (Emeryville, CA). Cell culture media, reagents, and bicinchoninic acid protein assay kit were purchased from Sigma (St. Louis, MO). The XTT cell proliferation kit was purchased from Roche (Basel, Switzerland). Horseradish peroxidase (HRP)-conjugated monoclonal antiphosphotyrosine antibody (4G10) was purchased from Upstate Biotechnology (Lake Placid, NY). Monoclonal anti-IGF1R antibody (IR-3) and polyclonal anti-IGF1R ß-subunit antibody (C-20) were obtained from Oncogene Research Products (Boston, MA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. GammaBind G Sepharose beads, the enhanced chemiluminescence detection kit, and donkey antirabbit IgGs coupled to HRP were purchased from Amersham Biosciences. Nitrocellulose membranes were purchased from Bio-Rad Laboratories (Hercules, CA). IGF1R clones (RP11 262P8 and RP11 34L10) were obtained from BACPAC Resources (Oakland, CA). The nick translation kit and the chromosome 15 control probe LSI D15S10 (which includes probes for the D15S10, D15Z1, and PML loci) were purchased from Vysis UK (Richmond, Middlesex, UK). [¹²⁵I]IGF-I was purchased from Amersham Pharmacia Biotech (Sunnyvale, CA).

Cytogenetics and fluorescence in situ hybridization (FISH) study

Routine karyotyping with Giemsa trypsin leishman banding was performed by standard techniques. The IGF1R clones (RP11 262P8 and RP11 34L10) were labeled by nick translation according to the manufacturer’s protocol (Vysis UK). FISH studies were undertaken with the IGF1R probes and the Vysis LSI D15S10 control probe also by standard techniques (12).

Monoclonal IGF1R and IR antibodies

The monoclonal IGF1R blocking antibodies, IGF1R 24–57 and IGF1R 24–60, and the monoclonal blocking antibody for IR, IR47–9, were obtained and characterized as described previously (13, 14, 15). To purify the mouse monoclonal antibodies from ascites fluids, an affinity chromatography kit (Mab Trap Kit, Amersham Biosciences) was used, and to change the buffer into PBS, dialysis slides (Pierce, Rockford, IL) were used.

Fibroblasts

Fibroblasts were cultured from skin biopsies. Fibroblast outgrowths were isolated and serially passed in Medium 199 supplemented with 10% fetal calf serum (FCS). Skin fibroblasts from normal donors were obtained from the National Institute of General Medical Sciences Human Genetic Cell Repository, Coriell Cell Repositories (Camden, NJ). The repository numbers and sex and age of the donors are as follows: GM 02036 (female, 11 yr) and AG 0795 (male, 2 yr). Fibroblasts from the patients and normal control cells were cultured in Medium 199 supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO₂ at 37 C.

Cell proliferation assay

Mitochondrial function was assessed using a previously described 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-{(phenylamino)carbonyl}-2H-tetrazolium hydroxide (XTT) assay (16). In this assay, the tetrazolium salt XTT is cleaved to an orange-colored formazan product by mitochondrial dehydrogenase in viable cells. Cells from the cases and normal control cells were plated into 96-wells (2500 cells per well) and incubated overnight in 100 µl/well of complete medium. Cells were then starved for 16 h in serum-free medium supplemented with 0.1% insulin-free BSA (Sigma) and 20 mM HEPES (pH 7.5). To obtain standard curves, cells were differentially diluted and treated as per the other plates. Cells were treated in the absence or presence of 10 µg/ml of receptor blocking antibody in serum-free medium for 1 h, followed by the addition of 10 nM rhIGF-I. The treatment was repeated daily. At the indicated periods of time, the medium was aspirated from the wells; 100 µl of DMEM without phenol red and 50 µl of XTT solution were added to each well. The cells were then incubated for 2–5 h at 37 C, and the absorbances in the wells were measured at 492 nm, corrected for the absorbances at 670 nm (Multiskan Ascent, Labsystems, Helsinki, Finland). All values from these assays were expressed as a percentage of those obtained from measurements of IGF-I-stimulated cellular proliferation without the presence of blocking antibodies.

Phosphorylation study

Cells were grown in 150-mm culture dishes in Medium 199 supplemented with 10% FCS until they reached confluence. Before growth factor stimulation, cultures of cells were switched to serum-free medium as described above for 16 h. Cells were then treated with 10 nM IGF-I in serum-free medium for 5 min at 37 C. The reactions were subsequently terminated by removing the medium and washing the cells twice with cold PBS. Cells were harvested with 700 µl lysis buffer [50 mM HEPES, 100 mM NaCl, 4 mM sodium pyrophosphate, 10 mM EDTA (pH 8), 1% Triton X-100, 10 nM sodium fluoride, 1 mM phenylmethylsulfonylfluoride, 2 mM sodium orthovanadate, 2 µg/ml leupeptin, and 2 µg/ml aprotinin]. After incubating at 4 C for 30 min, the lysates were subsequently centrifuged at 13,000 x g for 30 min at 4 C, and the supernatants were collected. Protein contents in the supernatants were determined by the bicinchoninic acid protein assay using BSA standards.

Each 1-mg aliquot of protein extracted from cultured cells was immunoprecipitated with 2 µg of monoclonal anti-IGF1R (IR-3) antibody and 40 µl of resuspended GammaBind G Sepharose beads overnight at 4 C. After washing the pellet three times with 1 ml of lysis buffer and once with 50 mM Tris (pH 8), the final pellet was resuspended in 30 µl of Laemmli buffer and boiled for 5 min. The immunoprecipitates were separated by 12% SDS-PAGE, under reducing conditions. The proteins were then electrophoretically transferred from gels to nitrocellulose membranes. Blots were exposed overnight to the HRP-conjugated antiphosphotyrosine (4G10) monoclonal antibody, diluted at 1:500 with 3% BSA/Tris-buffered saline-0.1% Tween 20. The antigen-antibody complexes were detected with the enhanced chemiluminescence detection kit. Membranes were visualized by exposure to BioMax film (Eastman Kodak Co., Rochester, NY). For analysis of IGF1R protein, the membrane was stripped and incubated with polyclonal anti-IGF1R ß-subunit antibody overnight at 4 C, followed by incubating with HRP-conjugated donkey antirabbit secondary antibody for 1 h at room temperature. Blots were quantitated by NIH image, and phosphorylation of IGF1R was normalized for IGF1R protein and expressed as a percentage of each unstimulated sample as a control.

Binding assay of IGF-I receptor

Binding studies were performed according to the method of Siebler et al. (17). Fibroblasts were seeded in 24-well plates (IWAKI, 2.5 x 10⁴ cells/well) and cultured in Medium 199 supplemented with 10% FCS (500 µl/well). After 48 h, cells were starved for 16 h. Quiescent cells were washed twice with 500 µl ice-cold Dulbecco’s PBS and incubated in 250 µl PBS containing 0.1% BSA with [¹²⁵I]IGF-I (23,000 cpm) in the presence or absence of 1 x 10^-11 to approximately 6.66 x 10^-8 M IGF-I for 24 h at 4 C. At the end of the incubation, cells were washed three times with 500 µl ice-cold PBS and solubilized by the addition of 250 µl 0.2 M NaOH. Radioactivity was measured in a -counter. Nonspecific binding was derived from the mean counts of three replicate wells in the presence of 6.66 x 10^-8 M IGF-I, and specific binding was determined by subtracting the nonspecific binding from the mean counts of three replicate wells.

Data analysis

Each experiment was performed at least three times. Calculations were performed using Excel 98 (Microsoft, Redmond, WA).

	Results

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Cytogenetics and FISH study

Case 1. Routine cytogenetic analysis showed a de novo terminal deletion of chromosome 15q26.1, karyotype 46,XX, del (15)(q26). FISH studies using the IGF1R probes confirmed deletion of the locus, with normal signal on the chromosome 15 homolog (Fig. 3A). Her FISH 22q11 testing was normal.

View larger version (44K): [in this window] [in a new window]   FIG. 3. Cytogenetics and FISH studies on the two cases. A, Case 1 with deletion of chromosome 15 (q26.1). Partial karyotypes of chromosome 15. Probes RP 11 262P8 and RP11 34L10 confirmed deletion of the IGF1R locus, with normal signal on one chromosome and absence of signal on the other chromosome homolog. B, Case 2 with duplication of one chromosome 15. Partial karyotypes of chromosome 15. FISH using probes RP11 262P8 and RP11 34L10 shows one signal on the normal chromosome and two signals on the duplicated chromosome. This patient has three copies of IGF1R rather than the usual two. The other signals on the chromosomes are the control probes D15Z1, D15S10, and PML (which map to p11.1, q11.2, and q22, respectively).

Cell proliferation assay

Cells were treated with 0.1–100 nM of IGF-I in serum-free medium. The maximal response was observed with IGF-I treatment at a dose of 10 nM. The different cell lines from normal controls showed similar cell proliferation, and therefore data were combined for analyses. In all cell types, IGF-I treatment stimulated cell proliferation (Fig. 4), and these effects were observed from d 2 through d 5. Overall effects were less than those seen with FCS. Cells from case 1 (IGF1R deletion) showed a significantly decreased response to 10 nM IGF-I when expressed as a percentage of control (mean ± 95% confidence interval) throughout the study period (d 2, 67.0 ± 17.0%; d 3, 63.3 ± 15.7%; d 4, 59.5 ± 7.9%; d 5, 57.0 ± 6.3%; all P < 0.0001). Proliferation in cells from this case was also reduced by d 5 when the cells were incubated without serum or IGF-I (Fig. 4A).

View larger version (15K): [in this window] [in a new window]   FIG. 4. The time course of mitochondrial activities as measured by the XTT assay. Results were expressed in terms of cell number/well using standard curves gained from each cell line [, , and • for cells from normal controls (GM 02036 and AG 0795), case 1, and case 2, respectively]. A, Starved cells in serum-free medium. B, Cells stimulated with 10% FCS. C, Cells stimulated with 10 nM IGF-I.

To assess whether the response to IGF-I was mediated via IGF1R or not, monoclonal blocking antibodies for IGF1R or IR were added 1 h before IGF-I treatment. Both IGF1R 24–60 and IGF1R 24–57 antibodies alone did not have any significant effects on cellular proliferation (data not shown). When these blocking antibodies were added with FCS, the IGF1R antibodies inhibited serum effects on cell proliferation by approximately 45–65% in all cell types (data not shown). When incubated with IGF-I, both IGF1R antibodies inhibited IGF-I stimulation of cell proliferation by 38–62% (Table 1), whereas the IR antibody did not.

Treatment with IGF-I for 5 min increased tyrosine phosphorylation of the IGF1R ß-subunit in all types of cells (Fig. 5). Basal and (10 nM) IGF-I stimulation of tyrosine phosphorylation in case 1 tended to be smaller relative to that in control cells when expressed as a percentage of control basal values (controls stimulated to 173 ± 54%; case 1 cells stimulated from 71 ± 36% to 105 ± 33%), but statistical significance was not reached. In cells from case 2, there was increased stimulation of tyrosine phosphorylation of the IGF1R ß-subunit by 10 nM IGF-I (113 ± 20% to 265 ± 36%; P < 0.05). IGF-I did not stimulate alterations in IGF1R protein expression in any of the cell types. IGF1R expression was significantly greater in cells from case 2 than in either control cells (P < 0.05) or cells from case 1 (P < 0.001) (when expressed as a percentage of control data, control 100 ± 8%; case 1, 72 ± 27%; case 2, 151 ± 21%). When the IGF1R phosphorylation was expressed relative to IGF1R protein, there was no significant difference between any of the cell types (IGF-I stimulated values, control, 190 ± 83%; case 1, 154 ± 14%; case 2, 150 ± 9%).

Binding assay of IGF-I receptor

Binding kinetics of [¹²⁵I]IGF-I to skin fibroblasts from case 1, case 2, and normal controls were investigated (Fig. 6, A and B). Scatchard plots demonstrated the similar affinity of IGF-I receptor to IGF-I among GM 2036, AG 0795, case 1, and case 2. However, the maximum number of binding sites (Bmax) in GM 2036, AG 0795, case 1, and case 2 was 9.94 ± 1.57 x 10⁶ sites/cell, 6.43 ± 3.29 x 10⁶ sites/cell, 4.00 ± 1.01 x 10⁶ sites/cell, and 11.54 ± 1.35 x 10⁶ sites/cell, respectively.

	Discussion

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We report two children, one short and one tall, who have one copy and three copies of the IGF1R gene, respectively. Growth rates of fibroblasts from these subjects, as assessed by mitochondrial activities stimulated by 10 nM IGF-I or 10% FCS, depended on the copy number of IGF1R gene. The stimulation of cell growth by IGF-I was at least partially inhibited by monoclonal anti-IGF1R -subunit antibodies, indicating that the accelerated growth by IGF-I was through the IGF1R. Our data indicate that in vitro mitochondrial activities on skin fibroblasts reflect IGF1R gene copy number and the clinical status. We also detected stronger expression of IGF1R protein and IGF-I-stimulated tyrosine phosphorylation of IGF1R in cells from the tall child with three copies of IGF-I gene. IGF-I binding study showed no significant differences in IGF-I affinity between cells from controls, case 1, and case 2. However, the maximum number of binding sites in the cells depended on copy number of the IGF1R gene.

The IGF1R gene has been assigned to 15q26.3. In the literature, several cases with deletion of the distal long arm of chromosome 15 with or without ring chromosome are described. Some reports included testing for an IGF1R gene deletion, and in all cases where the loss of one copy of the IGF1R gene was confirmed (17, 18, 19, 20, 21, 22, 23, 24, 25) there was IUGR and postnatal growth failure. Mild growth retardation is a common feature associated with any autosomal ring chromosome (26), but compared with children with ring chromosome 15 without IGF1R gene deletion, children who are hemizygous at the IGF1R locus have more severe growth failure (23), and these patients have other clinical features in common. All had some craniofacial abnormalities, including microcephaly, triangular facies, hypertelorism, high-arched palate, abnormal ears, and micrognathia, as well as skeletal abnormalities including clinodactyly, proximal placement of digits, club feet, and scoliosis. The clinical features overlap with Russell-Silver syndrome, although all those with deletions of 15q ter had developmental delay and some degree of mental retardation (10). IGF-I plays an important role in central nervous system development (27) as well as in cardiovascular development (28). Complex heart defects, also observed in some of those children, suggested the possibility of that hemizygosity for the IGF1R gene might be a risk factor for the development of cardiac abnormalities (25). Lack of one copy of the IGF1R gene might be expected to cause primary IGF-I resistance through a quantitative deficiency of the IGF1R. However, patients reported had normal GH levels and/or IGF-I levels in sera (17, 20, 22, 23).

The recurrent hypoglycemia in our patient with IGF1R gene deletion is difficult to explain. A child with short stature, recurrent hypoglycemia, and high levels of plasma GH and IGF-I has been reported previously (29). In that case, cytogenetic studies were not performed, but receptor binding studies with her skin fibroblasts showed a diminution of the specific binding of IGF-I by 50%. It is possible that the girl had a decreased number of IGF1R. Our case 1 had normal GH levels, and there was evidence of some spontaneous endogenous GH secretion even after GH therapy. It is possible that case 1 is IGF-I resistant, and this led to impaired GH suppression. Excess active IGF-I could bind to the IR causing hypoglycemia, but no data are available concerning her IGF-I levels on or off treatment.

Fibroblasts from case 1 showed reduced cell growth in response to IGF-I in vitro. Fibroblasts from two patients with deletion of the distal long arm of chromosome 15 showed approximately 50% decrease of the IGF1R mRNA expression, but normal IGF-I binding (17). They showed significant decreased biological response to IGF-I, compared with control cells, but when the data were expressed as net stimulation (maximal response minus basal), there was no significant decrease. Another study for IGF-I responsiveness in fibroblasts from children with short stature showed significantly reduced IGF-I sensitivity in cells from one patient (30). IGF1R expression in organs might be mosaic, and the study of cultured skin fibroblasts might not always reflect the situation in vivo.

Our case 1 has been treated with rhGH, and this led to some acceleration in growth that is compatible with our in vitro cell growth studies. There are a few case reports in the literature of children with similar defects treated with GH. A case with a ring 15 who did not have deletion of the IGF1R gene and whose IGF-I levels were subnormal was treated with GH, and they observed a good response to GH with sustained acceleration of growth and increase of IGF-I levels into the normal range (31). Another severely growth-retarded case with ring chromosome 15 and deletion of a single allele for the IGF1R gene was treated with rhIGF-I in graded doses of 40, 60, and 80 µg/kg twice daily by sc injection for periods of 2–2.5 d each (32). This short-term rhIGF-I treatment did not change mean GH level, but it increased mean IGF binding protein-3 level. In this patient, the in vitro studies showed a 50% reduction in IGF1R DNA and a quantitatively similar reduction in steady-state mRNA for IGF1R. In vitro binding of IGF-I to the patient’s fibroblasts was reduced, but the cells exhibited a growth response to the addition of IGF-I in a fashion similar to that of control fibroblasts (32).

We also report case 2 with tall stature and an extra copy of IGF1R gene. We believe that our case is the first case with three copies of IGF1R gene confirmed by FISH study. Since Parker and Alfi (33) reported the first case with partial trisomy of chromosome 15, several cases with a trisomy of the distal long arm of chromosome 15 were reported, but only a few cases have 15q26.3 duplication. One of the two girls with duplication 15q22-qter that Gregoire et al. (34) reported had a tall stature (>2 SD), as did two brothers with partial trisomy 15 (q25-qter): the older boy was 2.10 m in height at the age of 16 yr, and the younger one was 1.85 m tall at the age of 13 yr (35). A male case was reported who was trisomic for 15q24-qter, but the fetus died at the age of 15 wk having a size that corresponded to 18 wk (19). Two cases, partially trisomic for the distal part of the long arm of chromosome 15 (q21-qter) showed no growth retardation (36), but the other cases with partial trisomy of chromosome 15 had growth retardation, although not all the cases contain the region of 15q26 (37, 38, 39, 40, 41). Almost all reported cases with partial trisomy of chromosome 15 have learning difficulties and craniofacial dysmorphism (39, 40).

One of the problems in studies describing features of aneuploidy of a single gene is the difficulty of excluding other gene deletions/duplications that might contribute to the phenotype. This is particularly true for our patients, whose other phenotypic features such as delayed mental development, presumably result from the gain or loss of genes other than IGF1R from this region of chromosome 15. Both the deletion in case 1 and duplication in case 2 were cytogenetically visible. Estimated loss/gain of DNA in each case would be approximately 5 MB and would therefore involve several genes in addition to IGF1R. Nevertheless, our data indicate that copy number of the IGF1R gene in humans has a profound effect on prenatal and early postnatal growth. Relative IGF-I resistance or increased sensitivity in deletion and trisomy, respectively, explains the clinical features and is supported by in vitro studies.

View larger version (27K): [in this window] [in a new window]   FIG. 5. Cellular IGF-I-stimulated tyrosine phosphorylation and IGF1R expression. Cell lysates were immunoprecipitated with anti-IGF1R, and the immunoprecipitated proteins were probed with antiphosphotyrosine or anti-IGF1R antibodies. GM 02036 was used as control. IGF1R phosphorylation was normalized for IGF1R protein and expressed as a percentage of gain for each cell line compared to unstimulated lysate.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Specific fibroblast [125I]IGF-I binding. A, Displacement of [125I]IGF-I by increasing concentrations of unlabeled ligand. The binding assay was performed as described in Materials and Methods. Each point represents the mean [125I]IGF-I binding from three experiments. B, Scatchard plots of fibroblast [125I]IGF-I binding according to the method of Siebler et al. (17 ). IGF-I binding in each case was normalized for cell number. Each point represents the mean [125I]IGF-I binding from three experiments.

	Acknowledgments

	Footnotes

 
Y.O. was supported by a Traveling Fellowship from the Wellcome Trust (London, UK).

Abbreviations: FCS, Fetal calf serum; FISH, fluorescence in situ hybridization; HRP, horseradish peroxidase; IGF1R, type 1 IGF receptor; IR, insulin receptor; IUGR, intrauterine growth retardation; rhIGF-I, recombinant human IGF-I.

Received July 10, 2002.

Accepted September 11, 2003.

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Top Abstract Introduction Clinical Report Materials and Methods Results Discussion References

 

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